Purification of the mosquitocidal and cytolytic proteins of Bacillus thuringiensis subsp. israelensis

Abstract

Two proteins from parasporal crystals of Bacillus thuringiensis subsp. israelensis were purified to electrophoretic homogeneity by gel filtration and anion-exchange chromatography. The larger of the two proteins (molecular weight, 68,000) was not cytolytic, whereas the smaller protein (molecular weight, 28,000) was highly cytolytic when assayed against rat erythrocytes. When these proteins were assayed against larvae of the yellow fever mosquito, Aedes aegypti, the larger protein was at least 100-fold more toxic than the smaller protein. Although proteolytic activity was not detected in solubilized crystals nor in purified protein preparations, the toxin (molecular weight, 68,000) was readily degraded to smaller, nontoxic molecules, even when maintained at 4°C. Mixtures of the two purified proteins were significantly more toxic to mosquito larvae than was either protein alone. Thus, it is likely that both the mosquitocidal and the cytolytic protein play roles in the overall insecticidal action of the parasporal crystal produced by this bacterium.

BACILLUS THURINGIENSIS USE IN AGRICULTURE: A MOLECULAR PERSPECTIVE

“…toxins have also been shown to possess larvicidal activity against nematodes (Bone et al., 1988). T h e cryIV class of crystal protein genes (cryIVA, -B, -C, -D) and the cytA gene are located on the same plasmid and encode proteins with molecular weights in the range 28-140 kDa (Hurley et al, 1985;Insell & Fitz-James, 1985;Waalwijk et al, 1985; Lee, Eckblad & Bulla, 1985). T h e B.t.i.…”

“…T h e B.t.i. proteins are all synthesized at different times during sporulation, and are added to the developing inclusion in a step-wise manner (Lee et al, 1985). Minor differences in quoted protein sizes (molecular weights) arise from differences in the PAGE separation systems used by different laboratories.…”

The mode of action of Bacillus thuringiensis crystal proteins

“…Although Cyt proteins appeared toxic to various cell lines in vitro and to mice upon injection, crystals of mosquitocidal B. thuringiensis strains showed no in vivo toxicity to lepidopteran larvae when orally ingested. Several authors purified a 28 kDa protein from B. thuringiensis serotype israeIensis crystals, which appeared haemolytic but not toxic to Diptera (Hurley et al, 1985;Held etal., 1986;Visser etal., 1986;Wu & Chang, 1985). In contrast to these findings, others reported that extracts of recombinant E. coli cells expressing the CytA protein were hemolytic and slightly toxic to mosquitoes (McLean & Whiteley, 1987;Ward et al, 1984).…”

Toxicity of Bacillus thuringiensis variety israelensis against tipulid larvae

“…thuringiensis var. isruelensis strain IPS82 was allowed to sporulate in peptonized milk broth (HURLEY et al 1985) and crystals and spores were separated by CsCl gradient centrifugation as described reviously (VISSER et al 1986). The presence of spores in the crystal fraction was determinelby plate counting.…”